Original Article

Evaluation of Microbial Contamination of Dried Black (Gharahghorut) Produced in

Mojtaba Chizari ABSTRACT

(MSc) Department of Microbiology Background and objectives: Dried Black Curd (DBC), also and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, known as Gharahghorut (Persian), is a dairy product produced Shahid Beheshti University, Tehran, from curd of or doogh. The aim of this study was to Iran evaluate microbial contamination of industrially and traditionally Seyed Masoud Hosseini produced DBC in Iran. (PhD) Department of Microbiology and Microbial Biotechnology, Faculty Methods: Four DBC brands holding certification of the Institute of Life Sciences and Biotechnology, of Standards and Industrial Research of Iran (ISIRI), Iran Shahid Beheshti University, Tehran, Ministry of Health and Medical Education (IMHME) and ISO Iran were purchased from a market in Tehran, Iran. Microbial Ali Mohammadi monitoring for presence of coliform, Escherichia coli, coagulase- (PhD) Department of Microbiology, Faculty of Biological Sciences, positive Staphylococcus aureus, mold and yeast was performed Alzahra University, Tehran, Iran using different basic, enriched, selective and differential media Javad Fakhari including peptone water agar, MacCankey agar, blood agar and (MSc) Department of Microbiology brain heart infusion agar, etc. according to the ISIRI standards and Microbial Biotechnology, Faculty (No.13299). of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Results: None of the tested samples had microbial Iran contamination. However, in one traditional DBC sample, the Mojtaba Mashhadi Mohammadzadeh- Vazifeh population of mold and yeast was higher than the acceptable (MSc) Department of Microbiology level defined by the ISIRI standard. and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Conclusion: We found no contamination with coagulase- Shahid Beheshti University, Tehran, positive S. aureus, E. coli and coliforms in four brands of Iran traditionally and industrially produced DBC in Iran. This Corresponding author: Seyed Masoud Hosseini indicates that the hygienic practices designated by the IMHME

Email: [email protected] are well-implemented in the industrial sector. However, the

Tel: +9829905913 mold and yeast contamination in traditionally produced DBC

Address: Department of Microbiology should be prevented by applying hygienic practices during the and Microbial Biotechnology, Faculty process of manufacturing and distribution. of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Keyword: Dried black curd (DBC), Gharahghorut, Microbial Iran Contamination Received: 2020/04/24

Revised: 2020/06/14 Accepted: 2020/07/26

This work is licensed under a Creative Commons Attribution 4.0 License.

DOI: 0.29252/mlj.14.6.48

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INTRODUCTION However, ignoring food hygiene regulations Dried black curd (DBC), also known as can result in increased risk of DBC gharehghorut, gharaghorut, black kashk or contamination with foodborne pathogens, such qara, is a traditional product with black, as Escherichia coli, Staphylococcus aureus brown, white or yellow color and a very sour and molds. The present study aimed to assess taste. This stiff, sour and chewable dairy microbial contamination of traditionally and product is basically produced by boiling the industrially produced DBC in Iran. yogurt derived from mixture of kashk, MATERIALS AND METHODS and whey. It is used alone or along All reagents and microbial culture media with other raw materials for producing fruit used in the experiments were based on the leather. It is rich in lactic acid and contains recommendations of ISIRIs (No. 2461-1, some minerals such as calcium and vitamins, 2946, 6806-3 and 10899-2) (4–7). Sampling especially vitamin B2 (1). Based on the was performed based on the standard sampling preparation method provided in the Moeen protocol for agricultural products that are Encyclopedia, doogh is boiled and dried in a consumed orally (ISIRI No. 2836) (8). thick bag. Then, tuff or the remaining matter is Characteristics of DBC samples from shaped as bullets and further dried under manufacturers in Tehran (Iran) as well as the sunlight. Finally, the sour water extracted from type of certifications issued by different the bag is exposed to sunlight or boiled dry in authorities including the Iran Ministry of order to obtain gharaghorut. Health and Medical Education (IMHME), The hardness, softness, taste and color of DBC ISIRI, HACCP, ISO 9001 and ISO 22000 are depends on the amount and method of heating, summarized in table 2. Different DBC brands the level of fat, evaporated water, acidity and were purchased from a market in Tehran and sour. The composition of DBC is summarized were kept at 18-25 ˚C (Figure 1). in table 1. A healthy and standard DBC should The pH of DBC samples was determined and be free from mold and warehouse pests, microbial features were assessed using unpleasant and unnatural taste and smell, general, enriched, selective and differential external materials such as pebble, hair and media. In addition, presence of E. coli, burnt particles, color, additives and coagulase-positive S. aureus, coliforms, mold preservatives (1). and yeast was evaluated based on the protocols Gharaghorut is mainly produced traditionally provided by the ISIRI (No. 13299) (9). The at homes and less frequently in small factories. chemical composition of two DBC samples Accordingly, it is essential for producers to was analyzed. All measurements were carried receive an official hygiene code. Food out three times. spoilage can easily occur in the production Coliforms were isolated by using the most process, while physical, chemical and probable number (MPN) and colony counting biological contamination can have both health methods using enterobacteria enrichment and economic consequences. Spoilage also (EE) broth (Cat No. 651702, Quelab Inc., decreases the shelf life of the product and may Canada), violet red bile glucose agar (No. lead to food poisoning (2). Therefore, 651702, Quelab Inc., Canada) and nutrient microbial control of food products is a critical agar (Cat No. 105450, Merck, Germany) and o step to increase shelf life while maintaining incubation for 24 hours at 37 C. Glucose quality (3). The pH of DBC is low due to the oxidase fermentation test was conducted using high concentration of lactic acid, which limits glucose agar (Cat No. 651135, Quelab Inc., the growth of microorganisms. Canada) for further confirmation (ISIRI No. 11165 & 1,2-2461) (10–12).

Table 1. Sensory and physical characteristics of DBC based on the Institute of Standards and Industrial Research of Iran (ISIRI, No. 13299)

Indicator Description

Color The product should be uniform with a cream to dark blackish-brown color. Taste and smell DBC should possess sour taste without any unpleasant and unnatural smell. Consistency and The product should have a uniform texture in form of concentrated liquid to solid phase. texture

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Table 2. Characteristics of the purchased DBC brands

Sample code A B C D Number of sample Three Three Three Three Production method Industrial Industrial Traditional Traditional Color White Black White Black Hygiene code (IMHME)   x x

ISO 9001:2008 x x x x ISO 22000:2005 x x x x HACCP x x x x

Standard logo (ISIRI)   x x

Production date   x x Expiry date   x x

Note: the characteristics of samples A and B were written on their packages.

Figure 1. Color and appearance of the purchased DBC brands

Sample A Sample B

Sample C Sample D

The MPN technique and lauryl sulfate broth In addition, rabbit plasma (Cat No. 113306, (No. 652406, Quelab Inc., Canada), EC broth Merck, Germany) was used for confirmatory (Cat No. 110285, Merck, Germany) and test (ISIRI No. 3-6806) (13). The spread plate peptone water indole free (Cat No. 652106, technique was used to isolate and count molds Quelab Inc., Canada) were applied to isolate E. and yeasts. In this regard, samples were coli, which was confirmed by indole (ISIRI incubated in dichloran glycerol (DG18) agar (Cat No. 620238, Liofilchem, Italy) for 5-7 No. 2946) (6). Coagulase-positive S. aureus o were isolated using the MPN technique (5) and days at 25 C (ISIRI No. 2-10899) (7). After evaluating microbial contamination, the Giolitti-Cantoni broth (Cat No. 836930, obtained values were compared to standard Quelab Inc., Canada), Baird Parker agar (Cat levels (14). No. 105406, Merck, Germany) and brain heart The protein content of samples was infusion (BHI) broth (Cat No. 180239, Quelab determined via the Kjeldahl method based on Inc., Canada).

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the ISIRI standard (No. 1-9188) with standard to measure phosphorus level based on the acceptance level (SAL) of 8-12 (g/percent) ISIRI standard (No. 1808 (18). The method is (15). The fat content of samples was measured based on digestion of DBC samples in using the Soxhlet method based on the ISIRI sulphuric acid and hydrogen peroxide, then standard (No. 760) with SAL of ≤1 (g/percent) adding molybdate solution and ascorbic acid, (16). The ash content of samples was and finally measuring phosphate in the determined through ash analysis in a furnance solution at 820 nm (19). (550 oC) based on the ISIRI standard (No. RESULTS

1755) with SAL of ≤9 (g/percent) (17). Table 3 presents the results of culturing Moisture level was determined based on the DBC samples in culture media with different ISIRI standard (No. 1753) with SAL of 15-30 conditions. No microbial growth was observed (g/percent) (15). in culture of peptone water, plate count agar, The calcium content of DBC was determined MacConkey agar, blood agar, MRS agar, M17 using a Young Lin AAS 8020 atomic agar, nutrient agar, glucose agar, YM agar, absorption spectrometer (South Korea) with two phases, BHI agar, EMB and peptone water 99.7% burnt acetylene based on the ISIRI agar under different pH and aerobic/anaerobic standard (No. 9266). Moreover, the Unico UV conditions. / Vis 2100 spectrophotometer (USA) was used

Table 3. Results of culturing DBC samples in general, selective and differential media

Culture medium Culture condition Results of culturing DBC samples Sample A Sample B Sample C Sample D Peptone water Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND Plate count agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND MacConkey agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND Natural pH ND ND ND ND Blood agar pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND MRS agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND M17 agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND Nutrient agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND Glucose agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND YM agar Natural pH ND ND ND ND pH 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND Two phases Natural pH ND ND ND ND pH : 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND BHI agar Natural pH ND ND ND ND pH : 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND EMB Natural pH ND ND ND ND pH : 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND Peptone water agar Natural pH ND ND ND ND pH : 3 ND ND ND ND Anaerobic with natural pH ND ND ND ND Anaerobic with acidic pH ND ND ND ND

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In samples A and B that were holders of the They found contamination with E. coli and health code and ISIRI logo and other samples, coliform in 7% and 27% of the samples, no isolate was found in the tests related to respectively. However, there was no coliform, E. coli, coagulase-positive S. aureus, contamination with Salmonella and S. aureus mold and yeast. However, the number of (20). Unlike our study, the mentioned study molds and yeasts in samples C and D was assessed traditional DBC based on the ISIRI 1×102 and 3×103, respectively. standard for kashk not DBC. Table 5 shows the composition of an industrial The results indicated that the population of sample (A, white DBC) and a traditional mold and yeast in traditional DBC samples sample (C, white DBC). exceeded the standard level. However,

DISCUSSION implementing good hygiene practices in the As a traditional dairy product, DBC has traditional production process may been produced and consumed in various parts considerably reduce the extent of of Iran. Our study is the first to evaluate contamination. microbial contamination of both industrially The shelf life of DBC is long due to its low and traditionally produced DBC in Iran. This pH. Industrially produced DBCs had a two- dairy product is rich in protein and contains year expiry date. It should be noted that the high amounts of calcium, minerals and packages should be resistant to acid and vitamins. Thus, paying attention to this prevent the transfer of packaging materials to valuable product and improving its microbial the product, spoilage, contamination, drying or quality can be beneficial for the public health, exchange of moisture and smell (1). nutrition and development of the export sector. Furthermore, DBC should be kept in a cool, The results showed no microbial dry place and away from direct sunlight. The contamination in the tested samples of DBC. lack of microbial contamination in DBC The low microbial load can be caused by in- samples could also be due to the probable process high-temperature and repeated presence of growth inhibitors such as heating, very low pH and low percentage of bacteriophage and added materials containing active water and salt. Tajizadegan et al. antibiotics, residues of sterilizing agent and conducted a study on contamination of detergents, free fatty acids or substances traditional kashk and DBC with Salmonella, present in unprocessed food such as niacin as coliform, E. coli and S. aureus. well as natural antimicrobial proteins present in the DBC (21). Table 4. Indicator microbes’ counts with standard acceptance level (number/g) TEST NUMBER OF ISOLATED M ICROBES STANDARD ACCEPTANCE LEVEL (MPN/G) (NUMBER/GR) A B C D COLIFORM ND ND ND ND ≤ 10 (ISIRI No. 1 & 2-5486) E.COLI ND ND ND ND Negative (ISIRI No. 5234) COAGULASE-POSITIVE S. AUREUS ND ND ND ND Negative (ISIRI No. 3-6806) 2 MOLD AND YEAST ND ND 1×102 3×103 ≤ 10 (ISIRI No. 10154)

2 TOTAL COUNT OF ND ND 1×102 3×103 ≤ 10 (ISIRI No. 5484) MICROORGANISMS

ND: not detected

Table 5. Quantitative composition of samples A and C

Sample A C (g/percent) (g/percent)

Protein 8.40 5.16 Fat 0.40 0.30 Ash 11.90 8.10 Moisture 18.64 17.80 Dry matter 81.36 82.20 Calcium 1.70 1.95 Phosphorus 0.88 1.13

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The protein content of sample A (8.40 This indicates that the hygienic practices g/percentage) was at the allowed level, but the designated by the IMHME are well- protein content of sample C (5.16 implemented in the industrial sector. g/percentage) was lower than the standard However, the mold and yeast contamination in value defined by the ISIRI for DBC (8-12 traditionally produced DBC should be g/percentage). The difference in the protein prevented by applying hygienic practices content of the products could be related to the during the process of manufacturing and raw materials used in the traditional and distribution. industrial preparation of DBC. The fat content ACKNOWLEDGEMENTS of both sample A (0.40 g/percentage) and The authors would like to thank Research sample C (0.30 g/percentage) was at the and Development Department of Shahid allowed level compared to the standard value Beheshti University for the financial support. defined for DBC (max. 1 g/percent). The CONFLICT OF INTEREST moisture content of sample A and C was The authors declare that there is no determined as 18.64 and 17.80 g/percentage, conflict of interest. respectively, both of which were at the REFERENCES allowed level compared to the defined value 1. ISIRI (Institute of Standards and Industrial Research by the ISIRI for DBC (max. 15-30 g/percent). of Iran). Gharahghorut-Specifications and test methods. Therefore, DBC could be considered a healthy 2011;No:13299(ICS:67.100.99). dairy product with a long shelf life due to its 2. Smith JP, Daifas DP, El-Khoury W, Koukoutsis J, El- Khoury A. Shelf life and safety concerns of bakery low fat and moisture content compared to products—a review. Crit Rev Food Sci Nutr. 2004; other dairy products. Sample A and C 44(1): 19-55. doi: 10.1080/10408690490263774. included 11.90 and 8.10 g/percent of ash, 3. ISIRI (Institute of Standards and Industrial Research respectively. The ash content of sample A was of Iran). Microbiological of pastry and confectionary higher than the allowed value defined by the products-specifications and test method. 2019;No:2395:5–11. ISIRI standard (max. 9 g/percentage). Ash 4. ISIRI (Institute of Standards and Industrial Research represents the mineral content of DBC and its of Iran). Microbiology of food and animal feeding different values may be related to the level of stuffs-Horizontal methods for the detection and minerals consumed by the livestock whose enumeration of Enterobacteriaceae - Part 1: Detection and enumeration by MPN technique with per- milk was used for DBC preparation. The enrichment. 2007;No: 2461:1. phosphorus content of samples A and C was 5. ISIRI (Institute of Standards and Industrial Research determined as 0.88 and 1.13 g/percent, of Iran). Microbiology of food and animal feeding respectively. Moreover, the calcium content of stuffs-Horizontal method for the enumeration of samples A and C was determined as 1.70 and coagulase– positive Staphylococci (Staphylococcus aureus and other species) - Part 3: Detection and MPN 1.90 g/percent, respectively. Calcium is technique for low numbers. 2006;No: 6806-3. considered as the most abundant mineral in 6. ISIRI (Institute of Standards and Industrial Research the body, which is required for strengthening of Iran). Microbiology of food and animal feeding stuffs of bones and teeth, muscle contraction, -Detection and enumeration of presumptive Escherichia coli -Most probable number technique. 2005;No: secretion of specific hormones and proper 2946(ICS:30.100.07). functioning of some enzymes as well as the 7. ISIRI (Institute of Standards and Industrial Research nervous system. There are 125 mg of calcium of Iran). Microbiology of food and animal feeding stuffs in 100 g of low-fat milk and yogurt and the - Horizontal method for the enumeration of yeasts and value is lower in high-fat (4). molds - Part 2: Colony count technique in products with water activity less than or equal to 0.9. 2008;No: Considering the favorable calcium content of 10899-. DBC compared to that of milk and its 8. ISIRI (Institute of Standards and Industrial Research products, DBC can be considered as a of Iran). Packaged agricultural products used in food nutritious condiment and a suitable source of industry- Sampling. 2018; No: 2836(ICS: 67.040). 9. Mohammadi A, Hashemi M, Hosseini SM. calcium and other minerals for individuals Comparison of antifungal activities of various essential who do not consume milk or yogurt. oils on the Phytophthora drechsleri, the causal agent of

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How to Cite: This paper should be cited as: Chizari M, Hosseini SM, Mohammadi A, Fakhari J, Mashhadi Mohammadzadeh-Vazifeh M. [Evaluation of Microbial Contamination of Dried Black Curd (Gharahghorut) Produced in Iran]. mljgoums. 2020; 14(6): 48-54. DOI: 0.29252/mlj.14.6.48.

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